Methods for recombinant peptide production

ABSTRACT

The present invention provides improved methods for the production of recombinant peptides from bacterial cells.

This application is a continuation of U.S. Application No. 09/271,970,filed on Mar. 18, 1999 now U.S. Pat. No. 6,242,219.

The present invention relates generally to improved methods for theproduction of recombinant peptides from bacterial cells.

BACKGROUND OF THE INVENTION

Although bioactive peptides can be produced chemically by a variety ofsynthesis strategies, recombinant production of peptides, includingthose in the 50 amino acid size range, offers the potential for largescale production at reasonable cost. However, expression of very shortpolypeptide chains can sometimes be problematic in microbial systems,including in bacterial cells such as Escherichia coli. This is true evenwhen the peptide sequence is expressed as part of a fusion protein. Aspart of a fusion protein, peptides may be directed to specific cellularcompartments, i.e. cytoplasm, periplasm, or media, with the goal ofachieving high expression yield and avoiding cellular degradativeprocesses.

Preparation of a peptide from a fusion protein in pure form requiresthat the peptide be released and recovered from the fusion protein bysome mechanism and then obtained by isolation or purification. Methodsfor cleaving fusion proteins have been identified. Each methodrecognizes a chemical or enzymatic cleavage site that links the carrierprotein to the desired protein or peptide [Forsberg et al., I. J.Protein Chem. 11, 201-211, (1992)]. Chemical cleavage reagents ingeneral recognize single or paired amino acid residues which may occurat multiple sites along the primary sequence, and therefore may be oflimited utility for release of large peptides or protein domains whichcontain multiple internal recognition sites. However, recognition sitesfor chemical cleavage can be useful at the junction of short peptidesand carrier proteins. Chemical cleavage reagents include cyanogenbromide, which cleaves at methionine residues [Piers et al., Gene, 134,7, (1993)], N-chloro succinimide [Forsberg et al., Biofactors 2,105-112, (1989)] or BNPS-skatole [Knott et al., Eur. J. Biochem. 174,405-410, (1988); Dykes et al., Eur. J. Biochem. 174, 411-416, (1988)]which cleaves at tryptophan residues, dilute acid which cleavesaspartyl-prolyl bonds [Gram et al., Bio/Technology 12, 1017-1023,(1994); Marcus, Int. J. Peptide Protein Res., 25, 542-546, (1985)], andhydroxylamine which cleaves asparagine-glycine bonds at pH 9.0 [Moks etal., Bio/Technology 5, 379-382, (1987)].

Of interest is U.S. Pat. No. 5,851,802 which describes a series ofrecombinant peptide expression vectors that encode peptide sequencesderived from bactericidal/permeability-increasing protein (BPI) linkedvia amino acid cleavage site sequences as fusions to carrier proteinsequences. In some fusion protein constructs, an acid labileaspartyl-prolyl bond was positioned at the junction between the peptideand carrier protein sequences. BPI-derived peptides were released fromthe fusion proteins by dilute acid treatment of isolated inclusionbodies without prior solubilization of the inclusion bodies. Thereleased peptides were soluble in the aqueous acidic environment. Inaddition, BPI-derived peptides were obtained from fusion proteins underconditions where the fusion proteins were secreted into the culturemedium. Those secreted fusion proteins were then purified and treatedwith dilute acid to release the peptide.

Of additional interest are the disclosures of the following referenceswhich relate to recombinant fusion proteins and peptides.

Shen, Proc. Nat'l. Acad. Sci. (USA), 281, 4627 (1984) describesbacterial expression as insoluble inclusion bodies of a fusion proteinencoding pro-insulin and β-galactosidase; the inclusion bodies werefirst isolated and then solubilized with formic acid prior to cleavagewith cyanogen bromide.

Kempe et al., Gene, 39, 239 (1985) describes expression as insolubleinclusion bodies in E. coli of a fusion protein encoding multiple unitsof neuropeptide substance P and β-galactosidase; the inclusion bodieswere first isolated and then solubilized with formic acid prior tocleavage with cyanogen bromide.

Lennick et al., Gene, 61, 103 (1987) describes expression as insolubleinclusion bodies in E. coli of a fusion protein encoding multiple units(8) of α-human atrial natriuretic peptide; the inclusion bodies werefirst isolated and then solubilized with urea prior to endoproteinasecleavage.

Dykes et al., Eur. J. Biochem., 174, 411 (1988) describes solubleintracellular expression in E. coli of a fusion protein encoding α-humanatrial natriuretic peptide and chloramphenicol acetyltransferase; thefusion protein was proteolytically cleaved or chemically cleaved with2-(2-nitrophenylsulphenyl)-E-methyl-3′-bromoindolenine to releasepeptide.

Ray et al., Bio/Technology, 11, 64 (1993) describes solubleintracellular expression in E. coli of a fusion protein encoding salmoncalcitonin and glutathione-S-transferase; the fusion protein was cleavedwith cyanogen bromide.

Schellenberger et al., Int. J. Peptide Protein Res., 41, 326 (1993)describes expression as insoluble inclusion bodies of a fusion proteinencoding a substance P peptide (11a.a.) and β-galactosidase; theinclusion bodies were first isolated and then treated with chymotrypsinto cleave the fusion protein.

Hancock et al., W094/04688 (PCT/CA93/00342) and Piers et al. (Hancock),Gene, 134, 7 (1993) describe (a) expression as insoluble inclusionbodies in E. coli of a fusion protein encoding a defensin peptidedesignated human neutrophil peptide 1 (HNP-1) or a hybridcecropin/mellitin (CEME) peptide and glutathione-5-transferase (GST);the inclusion bodies were first isolated and then: (i) extracted with 3%octyl-polyoxyethylene prior to urea solubilization and prior to factorX_(a) protease for HNP1-GST fusion protein or (ii) solubilized withformic acid prior to cyanogen bromide cleavage for CEME-GST fusionprotein; (b) expression in the extracellular supernatant of S. aureus ofa fusion protein encoding CEME peptide and protein A; (c) proteolyticdegradation of certain fusion proteins with some fusion proteinpurified; and (d) proteolytic degradation of other fusion proteins andinability to recover and purify the fusion protein.

Lai et al., U.S. Pat. No. 5,206,154 and Callaway, Lai et al. Antimicrob.Agents & Chemo., 37:1614 (1993) describe expression as insolubleinclusion bodies of a fusion protein encoding a cecropin peptide and theprotein encoded by the 5′-end of the L-ribulokinase gene; the inclusionbodies were first isolated and then solubilized with formic acid priorto cleavage with cyanogen bromide.

Gramm et al., Bio/Technology, 12:1017 (1994) describes expression asinsoluble inclusion bodies in E. coli of a fusion protein encoding ahuman parathyroid hormone peptide and a bacteriophage T4-encoded gp55protein; the inclusion bodies were first isolated (6% wt/vol.) and thenwere treated with acid to hydrolyze the Asp-Pro cleavage site.

Kuliopulos et al., J. Am. Chem. Soc., 116:4599 (1994) describesexpression as insoluble inclusion bodies in E. coli of a fusion proteinencoding multiple units of a yeast α-mating type peptide and a bacterialketosteroid isomerase protein; the inclusion bodies were first isolatedand then solubilized with guanidine prior to cyanogen bromide cleavage.

Pilon et al., Biotechnol. Prog., 13, 374-379 (1997) describe solubleintracellular expression in E. coli of a fusion protein encoding apeptide and ubiquitin; the fusion protein was cleaved with a ubiquitinspecific protease, UCH-L3.

Haught et al., Biotechnol. Bioengineer., 57, 55-61 (1998) describeexpression as insoluble inclusion bodies in E. coli of a fusion proteinencoding an antimicrobial peptide designated P2 and bovine prochymosin;the inclusion bodies were first isolated and then solubilized withformic acid prior to cleavage with cyanogen bromide.

The above-references indicate that production of small peptides frombacteria has been problematic for a variety of reasons. Proteolysis ofsome peptides has been particularly problematic, even where the peptideis made as a part of a larger fusion protein. Such fusion proteinscomprising a carrier protein/peptide may not be expressed by bacterialhost cells or may be expressed but cleaved by bacterial proteases. Inparticular, difficulties in expressing cationic antimicrobial peptidesin bacteria have been described by Hancock et al. WO94/04688(PCT/CA93/00342) referenced above, due in their view to thesusceptibility of such polycationic peptides to bacterial proteasedegradation.

The production of peptide for preclinical and clinical evaluation oftenrequires multigram quantities [Kelley, Bio/Technology 14, 28-31 (1996)].If production of recombinant peptides can be achieved at this largescale, such production can potentially be economical. However,downstream processing steps for the production of peptides and proteinsfrom bacteria can often contribute a significant fraction of totalproduction cost. Initial recovery of peptide from bacterial inclusionbodies of fusion proteins, for example, generally requires multipledistinct processing steps, including the following four steps: (1) celldisruption/lysis, (2) isolation of inclusion bodies from thedisrupted/lysed cells, (3) solubilization of the isolated inclusionbodies in denaturant or detergent to obtain solubilized fusion protein,and (4) fusion protein cleavage and separation of peptide and carrierprotein. It is desirable that aspects of the recombinant productionprocess be improved and/or optimized in order to make large-scaleproduction of peptides by recombinant means more economically viable.

There continues to exist a need in the art for improved methods forrecombinant production of peptides from bacterial cells, particularlyfor simpler methods that do not require a multiplicity of steps,including, for example, the step of isolation or purification of peptidefusion proteins or the step of isolation or purification of inclusionbodies comprising the fusion proteins in order to obtain the recombinantpeptide.

SUMMARY OF THE INVENTION

The present invention provides improved methods for the production ofrecombinant peptides from bacterial cells. The improved methods precludethe need for the isolation and solubilization of inclusion bodies or theisolation and purification of peptide fusion proteins. The improvedmethods accomplish cell disruption/lysis and release of peptide frombacterial cells or bacterial cell cultures in a single step. Fusionproteins useful in methods of the invention comprise at least onepeptide sequence, a carrier protein sequence, and at least oneacid-sensitive amino acid cleavage site sequence located between thepeptide sequence and the carrier protein sequence. The inventionprovides improved methods for the microbial production of peptides fromsuch fusion proteins expressed intracellularly in bacterial cells. Therecombinant peptides recovered according to the invention are releasedby acid cleavage at the acid-sensitive cleavage site(s) in the fusionprotein. Recombinant peptides are thus efficiently and economicallyproduced according to the invention.

The invention thus provides an improved method for obtaining a peptidefrom bacterial cells after expression inside the cells of a fusionprotein, wherein the fusion protein comprises the peptide, a carrierprotein and an acid-cleavable site between the peptide and the carrierprotein, with the improvement comprising treating the bacterial cellswith acid under conditions sufficient in a single step to disrupt orlyse the cells and release the peptide from the fusion protein. Animproved method may include the additional step of obtaining thereleased peptide separated from the disrupted or lysed cells. Accordingto the invention, the released peptide may be separated from thedisrupted or lysed cells by a separation device, such as acentrifugation device or a filtration device. The invention alsoprovides an improved method for obtaining a peptide from bacterial cellsafter expression inside the cells of a fusion protein, wherein thefusion protein comprises the peptide, a carrier protein and anacid-cleavable site between the peptide and the carrier protein with theimprovement comprising the following steps: (a) treating the bacterialcells with acid under conditions sufficient to disrupt or lyse the cellsand release the peptide from the fusion protein; (b) separating solublematerial from insoluble material after step (a); and (c) recovering thereleased peptide in the soluble material after step (b). According tothe invention, the soluble material may be separated from the insolublematerial by a separation device, such as a centrifugation device or afiltration device. Improved methods of the invention may be employedwhere the bacterial cells are in cell culture media for the acidtreatment, or where the bacterial cells have been separated from cellculture media for the acid treatment, or where the bacterial cells arein cell culture media in a fermentation vessel for the acid treatment.According to methods of the invention, preferred acid-cleavable sites inthe fusion protein include an Asp-Pro cleavage site. Preferably, thecarrier protein is expressed as an insoluble protein inside thebacterial cells.

Improved methods of the invention for recombinant microbial productionof peptides from fusion proteins are based on the surprising discoverythat bacterial cell disruption/lysis and peptide release may beaccomplished simultaneously in a single step. According to theinvention, no process step is required for the isolation from the cellsof inclusion bodies and solubilization of such inclusion bodies prior topeptide release and recovery. Similarly, no process step is required forthe purification of the fusion proteins expressed in large amountsintracellularly as soluble or insoluble proteins in bacterial host cellsprior to peptide release and recovery. Remarkably, peptides efficientlyproduced as components of fusion proteins by the bacterial host cellsare efficiently cleaved and released from the fusion proteins by asingle step of cell disruption/lysis and peptide release. It isparticularly surprising that peptides according to the inventioneffectively made in E. coli are released in soluble form in this singlestep of cell disruption/lysis and peptide cleavage and are easilyrecovered from insoluble cell material. By way of example, recombinantBPI-derived peptides having one or more of the biological activities ofBPI (e.g., LPS binding, LPS neutralization, heparin binding, heparinneutralization, antimicrobial activity) have been produced and recoveredaccording to the methods of invention. Thus, the invention providesimproved methods of bacterial cell production of functional recombinantpeptides.

DETAILED DESCRIPTION

The present invention provides improved recombinant peptide productionmethods. Recombinant peptides encoded by and released from fusionproteins are recovered according to these improved methods. Fusionproteins useful in methods according to the invention comprise a peptidesequence, a carrier protein sequence and an acid-sensitive amino acidcleavage site sequence between the peptide and carrier proteinsequences. Improved methods according to the invention accomplish celldisruption/lysis and release of peptide from the cells in a single stepusing bacterial cells or bacterial cell cultures (e.g., fermentationcultures). The methods preclude the need for disruption/lysis followedby isolation and solubilization of inclusion bodies of the fusionproteins from the bacterial cells prior to peptide release and recovery.Unexpectedly, single step treatment of bacterial cells or bacterial cellcultures under conditions of acid pH and temperature sufficient tocleave and release peptides simultaneous with cell disruption/lysis,allows the direct recovery of soluble peptide from insoluble cell lysismaterial. Fusion proteins containing BPI-derived peptides withanti-microbial activity were expressed intracellularly in large amountswithout significant proteolysis, until acidification of the bacterialcells. A variety of BPI-derived peptides, including those comprising thesequences listed in Table 4 of U.S. Pat. No. 5,851,802 incorporated byreference herein in its entirety, may be produced by recombinant methodsaccording to the invention.

An advantage provided by the present invention is the ability to producepeptides from fusion proteins more efficiently and economically frombacterial host cells. Additional advantages include the ability torecover and obtain homogeneous peptide in large amounts via improvedmethods that are particularly amenable to scale-up in large fermentationvessels. “BPI-derived peptide” or “BPI peptide” as used herein refers toa peptide derived from or based on bactericidal/permeability-increasingprotein (BPI), including peptides derived from Domain I (amino acids17-45), Domain II (amino acids 65-99) and Domain III (amino acids142-169) of BPI (SEQ ID NOS: 15 and 16), each peptide having an aminoacid sequence that is the amino acid sequence of a DPI functional domainor a subsequence thereof and variants of the sequence or subsequencehaving at least one of the biological activities of BPI. The amino acidsequence of the entire human BPI protein and the nucleic acid sequenceof DNA encoding the protein have been reported in FIG. 1 of Gray et al.,J. Biol Chem., 264, 9505 (1989), incorporated herein by reference. TheGray et al. DNA and amino acid sequences are set out in SEQ ID NOS: 15and 16 hereto. An N-terminal BPI fragment of approximately 23 kD,referred to as rBPI₂₃, [Gazzano-Santoro et al., Infect. Immun. 60,4754-4761 (1992)], an analog designated rBPI₂₁ or rBPI₂₁Δcys (U.S. Pat.No. 5,420,019, incorporated by reference herein) as well as recombinantholoprotein, also referred to as rBPI, have been produced havingsequences set out in SEQ ID NOS: 15 and 16, except that valine atposition 151 is specified by GTG rather than GTC and residue 185 isglutamic acid (specified by GAG) rather than lysine (specified by AAG).As used herein, a “biological activity of BPI” refers to LPS binding,LPS neutralization, heparin binding, heparin neutralization orantimicrobial activity (including anti-bacterial and anti-fungalactivity). Such BPI-derived peptides having at least one of theactivities of BPI may be useful as antimicrobial agents (includinganti-bacterial and anti-fungal agents), as endotoxin binding andneutralizing agents, and as heparin binding and neutralizing agentsincluding agents for neutralizing the anticoagulant effects ofadministered heparin, for treatment of chronic inflammatory diseasestates, and for inhibition of normal or pathological angiogenesis.“Cationic BPI peptide” refers to a BPI peptide with a pI>7.0.

As used herein a “transformed bacterial host cell refers to a bacterialcell that contains recombinant genetic material or a bacterial cell thatcontains genetic material required for expression of a recombinantproduct. The genetic material may be introduced by any method known inthe art including transformation, transduction, electroporation andinfection.

As used herein, a “vector” or “vector construct” refers to plasmid DNAthat contains recombinant genetic material which may encode arecombinant product(s) and may be capable of autonomous replication inbacteria.

“Carrier protein” as used herein refers to a protein that can beexpressed in bacteria and used as a fusion partner to a linked peptideor protein. Preferred carrier proteins are those that can be expressedat high yield and when used as a fusion partner can confer highlevel-expression to a linked peptide or protein. Particularly preferredcarrier proteins are those that are expressed intracellularly as solubleor insoluble proteins, such as the D subunit of a human osteogenicprotein (“Bone D”). Any known carrier protein may be utilized as aprotein fusion partner, including, for example, ubiquitin, [see e.g.,Pilon et al., Biotechol. Prog. 13, 374-379 (1997)]; staphylococcalprotein A, [see e.g., Uhlén et al., Gene 23, 369:378 (1983) and Piers etal., Gene 134, 7-13 (1993)]; thioredoxin, [see e.g. LaVallie et al.,Bio/Technology 11, 187-193 (1993)]; maltose binding protein, [see e.g.,Tsao et al., Gene 169, 59-64 (1996)]; glutathione-s-transferase, [seee.g., Ray et al., Bio/Technoloty 11 64-70 (1993) and Piers et al., Gene134, 7-13 (1993)]; prochymosin, [see e.g. Haught et al., Biotechnologyand Bioengineering 57, 55-61 (1998)]; β-galactosidase, [see e.g., Kempeet al., Gene 39, 239-245 (1985)]; and gp 55 from T4, [see e.g., Gram etal., Bio/Technology 12, 1017-1023 (1994)]. A “cationic carrier protein”as used herein refers to a carrier protein having a pI (as calculatedbased on amino acid sequence or as measured in solution) greater than7.0 and preferably greater than 8.0. Such proteins include (1) Bone D(pI 8.18) (SEQ ID NOS: 1 and 2) and (2) gelonin (pI 9.58) (see, e.g.,U.S. Pat. Nos. 5,416,202 and 5,851,802, hereby incorporated by referencein their entirety).

“Amino acid cleavage site” as used herein refers to an amino acid oramino acids that serve as a recognition site for a chemical or enzymaticreaction such that the peptide chain is cleaved at that site by thechemical agent or enzyme. Amino acid cleavage sites include those ataspartic acid-proline (Asp-Pro), methionine (Met), tryptophan (Trp) orglutamic acid (Glu). “Acid-sensitive amino acid cleavage site” as usedherein refers to an amino acid or amino acids that serve as arecognition site such that the peptide chain is cleaved at that site byacid. Particularly preferred is the Asp-Pro cleavage site which may becleaved between Asp and Pro by acid hydrolysis.

Peptides derived from or based on BPI (BPI-derived peptides), aredescribed in co-owned U.S. Pat. No. 5,858,974 [WO 97/04008(PCT/US96/03845)]; U.S. patent application Ser. Nos. 08/504,841 and09/119,858 [WO 96/08509 (PCT/US95/09262)]; U.S. Pat. Nos. 5,652,332 and5,856,438 [WO 95/19372 (PCT/US94/10427)]; U.S. Pat. Nos. 5,733,872 and5,763,567 [WO 94/20532 (PCT/US94/02465)]; U.S. Pat. Nos. 4,348,942;5,639,727; 5,807,818; 5,837,678; and 5,854,214 [WO 94/20128(PCT/US94/02401)]; the disclosures of all of which are incorporatedherein by reference.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples whereinExample 1 addresses construction of fusion protein expression vectorconstructs; Example 2 addresses expression of recombinant fusionproteins; Example 3 addresses acid hydrolysis of bacterial cells orbacterial cell cultures and release of recombinant peptide; Example 4addresses acid hydrolysis of bacterial cell cultures in fermentationvessels; Example 5 addresses acid hydrolysis of bacterial cells afterremoval of cell culture medium; Example 6 addresses recovery andpurification of recombinant peptides from acid hydrolyzed bacterialcells; and Example 7 addresses biological activity assays of recombinantpeptides.

EXAMPLE 1 Construction of Fusion Protein Expression Vectors

1. Bacterial Expression Vector Construct pING4702

A bacterial expression vector which would encode a peptide fusionprotein, was constructed. This vector contains a sequence for a geneencoding subunit D of a human osteogenic protein (“Bone D”) (see, aminoacids 23 through 161 of SEQ ID NOS: 1 and 2), linked to a sequenceencoding a linking sequence that includes the dipeptide Asp-Pro and asequence encoding a peptide derived from the sequence of BPI (SEQ ID NO:3). This vector construct, pING4702, was prepared in several steps asdescribed below.

First, two synthetic oligonucleotides were synthesized that encode aBPI-derived peptide, an Asp-Pro dipeptide and appropriate restrictionenzyme recognition sites for cloning. The oligonucleotides encoding thissequence were:

5′-GATCCACCGAAAGTGGGTTGGCTGATCCAGCTGTTCCACAAAA AGTAAAGC-3′ (SEQ ID NO:4)

5′-TCGAGCTTTACTTTTTGTGGAACAGCTGGATCAGCCAACCCACTTT CGGTG-3′ (SEQ ID NO:5)

Sixteen μg of each oligonucleotide were annealed in a 50 μL reaction in100 mM NaCl, 10 mM Tris, pH 7.8, 1 mM EDTA for 10 minutes at 68° C., 30minutes at 57° C., and followed by slow cooling to room temperature. Theresulting annealed oligonucleotide fragment encodes an Asp-Pro-Prosequence followed by sequence encoding a peptide with the 12 amino acidsequence of XMP.391, as described in U.S. Pat. No. 5,851,802: Asp ProPro Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys (SEQ ID NO: 6)

The annealed oligonucleotide fragment also contains restriction enzymesites for cleavage by BamHI at the 5′ end and XhoI at the 3′ end ofsequence. The resulting annealed oligonucleotide was purified bycentrifugation on a Chroma Spin 10 column (Clontech, Palo Alto, Calif.).

Second, DNA fragments from two plasmid vectors were prepared. PlasmidpIC100, a derivative of pBR322 and which includes the leader sequence ofthe E. carotovora pelB gene, described in U.S. Pat. No.5,416,202 (see,e.g., Example 10) incorporated by reference, was digested with EcoRI andXhol, and the large vector fragment of approximately 2836 bp, waspurified. Plasmid pING3353, described in U.S. Pat. No. 5,851,802,incorporated by reference, was digested with EcoRI and BamHI and theapproximately 550 bp fragment which encodes the peIB:Bone D protein waspurified.

Third, the annealed oligonucleotide, the EcoR1 to XhoI fragment frompING100 and the EcoRI to BamHI fragment from pING3353 were ligated in 20μL 50 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT, 5% PEG-8000 with3 Units T4 DNA Ligase for 16 hours at 4C° to generate the intermediatevector pING4700. Plasmid pING4700 confers ampicillin resistance andencodes the fusion protein Bone D-Asp-Pro-peptide.

Plasmid pING4700 was digested with EcoRI and AhoI, and the 604 bpfragment encoding the fusion protein was ligated to the approximately5500 bp vector fragment from pING3217, as described in U.S. Pat. No.5,851,802, (see Example 1), that had been digested with EcoRI and AhoIin μL 50 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT, 5% PEG-8000with 3 Units T4 DNA Ligase for 16 hours at 4C°. The resulting plasmid,pING4702, encodes the Bone D-Asp-Pro-Pro-peptide fusion protein (SEQ IDNOS: 7 and 8) under the transcriptional control of the araB promoter.Plasmid pING4702 confers resistance to the antibiotic tetracycline.

2. Bacterial Expression Vector Construct pING4703

A second bacterial expression vector was constructed which encodes apeptide fusion protein containing Bone D (see, amino acid 23 through 161of SEQ ID NOS: 1 and 2), the dipeptide Asp-Pro and a 25 amino acidpeptide derived from the sequence of BPI (SEQ ID NO: 9). This vectorconstruct, pING4703, was prepared as described below.

First, two synthetic oligonucleotides were synthesized that encode aBPI-derived peptide, an Asp-Pro-Pro sequence and appropriate restrictionenzyme recognition sites for cloning. The oligonucleotides encoding thissequence were:

5′-CATTGGATCCACCGAAATGGAAGGCCCAGTTTCGCTTTCTTAA GAAA TCGAAAGTGGGTTG-3′(SEQ ID NO: 10)

5′-GGCTCTCGAGCTCTACTTTTTATGAAACAGCAGGATCAGCCAACC CACTTTCGATTTCTTA -3′(SEQ ID NO: 11)

Sixteen μg of each oligonucleotide were annealed in a 50 μL reaction in100 mM NaCl, 10 mM Tris, pH 7.8, 1 mM EDTA for 10 minutes at 68° C., 30minutes at 57° C., followed by slow cooling to room temperature. Analiquot of the annealed oligonucleotides was diluted into 10 mM Tris, pH8.3, 50 mM KCl (300 μL total volume) and filled-in with a reactioncontaining AmpliTaq (Perkin Elmer, Norwalk, Conn.), dATP, dGTP, dCTP anddTTP at 72° C. The resulting double-stranded fragment encoded therestriction sites BamHI and XhoI at the 5′ and 3′ ends, respectively,and encoded an Asp-Pro-Pro sequence followed by a sequence encoding apeptide with the 24 amino sequence of XMP.102, as described in U.S. Pat.No. 5,851,802:

Asp Pro Pro Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys Val GlyTrp Leu Ile Leu Leu Phe His Lys Lys (SEQ ID NO: 12)

The double-stranded fragment was digested with BamHI and Xhol, andligated to both the approximately 5500 bp EcoRI to XhoI vector fragmentfrom pING3217, and the approximately 550 bp EcoRI to BamHI fragment ofpING3353 in 20 μL 50 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM ATP, 1 mMdithiothreitol, 5% PEG-8000 with 1 Unit T4 DNA Ligase for 16 hours at4C°. The resulting plasmid, pING4703, encodes the BoneD-Asp-Pro-Pro-peptide fusion protein (SEQ ID NOS: 13 and 14) under thetranscriptional control of the araB promoter. Plasmid pING4703 confersresistance to the antibiotic tetracycline.

EXAMPLE 2 Expression of Recombinant Fusion Proteins

Expression of a recombinant product under control of the araB promoterwas evaluated as follows. Expression vector constructs were transformedinto E. coli E104 (deposited as ATCC 69009; ATCC 69008; ATCC 69101; ATCC69102; ATCC 69103; ATCC 69104; ATCC 69331; ATCC 69332; ATCC 69333, eachcontaining a gelonin-encoding plasmid) and tetracycline resistantcolonies were selected. Bacterial cultures from these colonies weregrown at 37° C. in TYE medium (15 g Tryptone, 10 g Yeast Extract, 5 gNaCl per liter) supplemented with 15 μg/mL of tetracycline. For storageof bacterial cells prior to growth in a fermentor, bacterial cultures (1to 2 mL) were frozen in TYE medium supplemented with 15% glycerol andstored at −20° C. To initiate production of recombinant product, a vialof cells containing the product expression vector was thawed, andinoculated into 100 mL of GMM culture medium as described below andgrown to approximately 200 Klett Units, then inoculated into either a 14L or 35 L fermentor. Each fermentor contained a minimal salts mediumwith glycerol as a carbon source (Glycerol Minimal Medium, GMM). The 14L or 35 L fermentor vessel initially contained approximately 7 L or 20L, respectively, of GMM which contains the following ingredients perliter:

Autoclaved Ingredients (NH₄)₂SO₄ 12 g KH₂PO₄ 1.57 g K₂HPO₄ 14.1 gMgSO₄.7H₂O 0.28 g H₃PO₄ (Conc.) 3 mL Antifoam 1 mL Biotin 0.0012 g YeastExtract 4.6 g Glycerol 18.5 g Filter sterilized ingredients CaCl₂.2H₂O(10% w/v) 1 mL Trace D Solution* 16 mL Thiamine HCl (10% w/v) 0.1 mLNicotinic Acid (1% w/v) 2 mL *Trace D solution is composed of:FeCl₃.6H₂O 6.480 g ZnSO₄.7H₂O 1.680 g MnCl₂.4H₂O 1.200 g Na₂MoO₄.2H₂O0.576 g CuSO₄.5H₂O 0.240 g CoCl₂.6H₂O 0.240 g H₃BO₃ 0.720 g H₃PO₄(Conc.) 96.0 mL H₂O (Batch Volume) 2.0 L

The fermentor was then inoculated with the bacterial seed culture, andwas maintained at pH 6.0 and 32° C. with 10 L/min. air and agitation at1000 rpm. When nutrients became limiting (as judged by an increase inthe dissolved oxygen, DO, to approximately 100%), the culture was fedwith additional nutrients until the culture reached an optical density(OD₆₀₀) of approximately 100. Culture feed rate was controlled tomaintain the DO to a setpoint of 20%. Specifically, the culture was fedwith the first feed:

Autoclaved ingredients per liter of feed: Glycerol 700 g MgSO₄.7H₂O 10 gBiotin 0.01 g Filtered ingredients per liter of feed CaCl₂.2H₂O (10%w/v) 35 mL Thiamine HCl (10% w/v) 3.5 mL Nicotinic Acid (1% w/v) 7 mL

The culture was induced by gradient induction at an OD of approximately100 with a second feed containing the inducing agent L-arabinose.Specifically, the second feed was:

Autoclaved ingredients per liter of feed: Glycerol 700 g MgSO₄.7H₂O 10 gBiotin 0.01 g Arabinose 60 g/L Filtered ingredients per liter of feedCaCl₂.2H₂O (10% w/v) 35 mL Thiamine HCl (10% w/v) 3.5 mL Nicotinic Acid(1% w/v) 7 mL

The cultures were harvested 23-26 hours post induction.

The cells may be separated from the culture medium with a 0.2 μm hollowfiber cartridge, 10 ft.² (Microgon, Laguna Hills, Calif.) as describedin Examples 3 and 5 below. Alternatively, the fermentation broth (i.e.,culture medium with cells) may be used directly in the fermentationvessel or removed from the fermentor for acidification and furtherprocessing as described in Examples 4 and 5 below. Example 6 belowdescribes the recovery and purification of recombinant peptides.

EXAMPLE 3 Acid Hydrolysis of Bacterial Cells and Release of RecombinantPeptide

1. Peptide Release from Bacterial Cells

Previously, inclusion bodies were isolated. Acid treatment of theisolated inclusion bodies resulted in the hydrolysis of theaspartyl-prolyl bond (Asp-Pro) between the Bone D protein and arecombinant peptide by incubation in dilute acid at elevatedtemperatures (see, e.g. Example 3 of U.S. Pat. No. 5,851,802). In theseexperiments, bacterial cells were directly acidified in an attempt tolyse the cells and hydrolyze the inclusion bodies directly to releasethe peptide. This was done by diluting cells in dilute acid at elevatedtemperature. E. coli E104 containing plasmid pING4702 was grown in a 10L fermentor and induced with arabinose. After termination of thefermentor run, bacterial cells were separated from the majority of theculture supernatant with a 0.2 μm hollow fiber cartridge (Microgon,10ft²) and frozen. Cells obtained from the fermentor were thawed andincubated under acidic conditions for 4 hours at 85° C. as follows inTable 1.

TABLE 1 Sample Cell Paste Incubation Condition A 1 gram 10 mL of 30 mMHCl B 1 gram 10 mL of 30 mM HCl, 5 mM EDTA C 1 gram 10 mL of 30 mM HCl,5 mM EDTA, 1% Triton X-100 D 1 gram 10 mL of 30 mM HCl, 5 mM EDTA, 8 Murea

As a control, approximately 1 gram of cells were lysed with lysozyme andinclusion bodies were isolated prior to acid hydrolysis according toprior methods (see, e.g., Example 3 of U.S. Pat. No. 5,851,802). Thesecells were suspended in 10 mL of 100 mM Tris, 5 mM EDTA, pH 8.0. Theslurry was incubated on ice for 15 minutes, and 1 mL of 10 mg/mLlysozyme was added and incubated on ice for 20 minutes. To disrupt thelysozyme treated cells, the slurry was sonicated 4 times for 10 secondseach at the highest setting using a Sonic U sonicator (B. Braun BiotechInc., Allentown, Pa.). The lysed cells were centrifuged at 13,000 rpm ina JA20 rotor for 25 minutes. The inclusion body pellet was thenincubated in 10 mL of 30 mM HCl for 4 hours at 85° C.

Prior to incubation at 85° C., the pH of all samples was adjusted to pH2.5 with HCl, except for the sample containing urea which was adjustedto pH 3.0. After incubation, the samples were centrifuged at 13,000 rpmin a JA20 rotor for 25 minutes to separate soluble from insolublematerial. The amount of released peptide in the supernatant from eachsample was evaluated by HPLC using a Beckman Coulter (Fullerton, Calif.)instrument with a Shimadzu Scientific Instruments (Columbia, Md.) autoinjector and a Vydac (Hesperia, Calif.) C18 (#218TP54) column. Solvent Awas 10% acetonitrile/0.1% TFA; solvent B was 90% acetonitrile/0.1 % TFA.The column was run with an 20-40% B gradient over 20 minutes at a flowrate of 1 mL/minute with peptide detection at 229 nm.

The concentration of peptide in the supernatant was as follows in Table2.

TABLE 2 Concentration % of Sample (mg/mL) Control Purified Inclusionbodies (Control) 0.292 100 A 10 mL of 30 mM HCl 0.223 76.4 B 10 mL of 30mM HCl, 5 mM EDTA 0.218 74.7 C 10 mL of 30 mM HCl, 5 mM EDTA, 1% 0.28798.3 Triton X-100 D 10 mL of 30 mM HCl, 5 mM EDTA, 8 0 0 M urea

These data demonstrate for the first time that peptide could be releaseddirectly from cells by incubation of the bacterial cells in dilute acidwhile the majority of other proteins remain insoluble.

2. Timecourse of Peptide Release from Bacterial Cells.

The results described above demonstrated that peptide was released froma Bone D-peptide fusion protein containing an acid sensitive Asp-Propeptide linker by direct hydrolysis of cells in dilute acid. Studieswere performed to examine the timecourse for hydrolysis. A sample of thesame concentrated, frozen cell sample described above was used foradditional studies. Approximately 2 grams of cell paste was diluted with20 mL of water and concentrated HCl was added to bring the pH to 2.5.The sample was incubated at 85° C., and samples were removedperiodically for quantitation. Each sample was centrifuged to removeinsoluble material, and the supernatant was assayed for released peptideby HPLC. The concentration of peptide in the soluble fraction was asfollows in Table 3.

TABLE 3 Concentration by HPLC Time (Hours) (mg/mL) 0 0 0.5 0.02 1 0.03 20.056 3 0.082 4 0.142 5 0.184 6 0.219 7 0.267

Thus, as shown in Table 3, the amount of peptide in the soluble fractionwas still increasing at the end of the seven hour timecourse.

In additional studies, a cell sample of bacterial cells in cell culturemedia was incubated at pH 2.15 to evaluate the timecourse of peptiderelease from cells that had not been previously concentrated and frozen.Specifically, 40 mL of bacterial cells in fermentation broth(fermentation culture of E. coli E104 containing pING4702) at the end ofthe fermentor process as described in Example 2 was directly adjusted topH 2.15 by adding 500 μL of concentrated HCl, to a final concentrationof approximately 150 mM. The sample was incubated at 85° C. and everyhour a sample was removed, centrifuged, and the supernatant wasevaluated for peptide by HPLC. The amount of peptide released over timewas as follows in Table 4.

TABLE 4 Concentration by HPLC Time (Hours) (mg/mL) 0 0 1 0.062 2 0.218 30.321 4 0.350 5 0.366 6 0.392 7 0.432 8 0.401 23 0.298

At pH 2.15 and using cells directly in the fermentation medium, maximumrelease peptide occurred by seven hours at 85° C., after which theamount of released peptide decreased.

Additional studies demonstrated that dilute H₂SO₄ and HNO₃ could alsorelease soluble peptide from bacterial cells and bacterial cells in cellculture media (i.e., fermentation cultures). In studies with H₂SO₄, two20 mL samples of bacterial cells in fermentation broth as described inExample 4 (fermentation culture of E. coli E104 containing pING4702)were collected after completion of bacterial fermentation, and they wereacidified to pH 2.4. One sample was adjusted to pH 2.4 with HCl and theother was adjusted to pH 2.4 with H₂SO₄. Each sample was incubated at85° C., a sample was removed every hour for seven hours and the amountof soluble peptide in each sample was analyzed by HPLC. Theconcentration of peptide in each aliquot is shown in the following Table5.

TABLE 5 Peptide Concentration by HPLC (mg/mL) Sample Time (Hour) HClHydrolysis H₂SO₄ Hydrolysis 0 0 0 1 0.034 0.036 2 0.130 0.116 3 0.1850.162 4 0.231 0.209 5 0.281 0.248 6 0.295 0.275 7 0.303 0.273

In studies with HNO₃, a sample of cells in bacterial fermentation brothas described in Example 4 was incubated with nitric acid. Specifically,20 mL of cells were adjusted to pH 2.2 with nitric acid and incubated at85° C. Samples were removed periodically and the concentration ofrecombinant peptide in the soluble fraction was determined by HPLC. Theconcentration of peptide in each aliquot is shown in the following Table6.

TABLE 6 Peptide Concentration by HPLC (mg/mL) after Sample Time (Hour)Hydrolysis with HNO₃ 0 0 1 0.093 2 0.146 4.5 0.308 6 0.325

These additional studies demonstrate that acids such as nitric acid,that are less corrosive to stainless steel materials used infermentation vessels, are useful in the improved methods of theinvention.

EXAMPLE 4 Acid Hydrolysis of Bacteria Directly in a Fermentation Vessel

Since peptide was released from bacterial cells and cell cultures bydirect incubation of cells in acid as described in Example 3, studieswere done to evaluate if soluble peptide could be recovered directlyfrom a bioreactor at the end of the fermentation process when contentsof the fermentor were acidified and heated in place. In initial studies,E. coli E104 containing pING4702 was grown in a 35 L fermentor asdescribed in Example 2. The first feed solution was introduced in thefermentor at 20.5 hours after inoculation, and the culture was inducedwith the second feed when the OD600 had reached 97.2. At 62.5 hoursafter induction, 10% HCl was added to the fermentor in 50 mL aliquotsuntil the pH of the fermentor had reached approximately 2.28. In total,990 mL of acid was added to the approximately 26 L of fermentationproduct in the fermentor. After reducing the pH, the temperaturesetpoint on the fermentor was increased to 85° C., and samples wereremoved from the fermentor periodically thereafter for six hours. Thecontents of the vessel were mixed during the reaction with the fermentorimpellers. BPLC analysis of the soluble material in the samples revealedthat the concentration of the peptide leveled off between four and fivehours. The concentration of peptide was as shown in the following Table7.

TABLE 7 Sample Timepoint (Hours) Peptide Concentration by HPLC (mg/mL) 00 1 0.185 2 0.294 3 0.334 4 0.353 5 0.353 6 0.370

In additional studies, E. coli E104 containing pING4702 was grown in a35 L fermentor to an OD600 of 89, induced with the second feedcontaining arabinose and grown for 24 hours. A 10% HCl solution wasadded to bring the culture pH to approximately 2.3, and the temperaturewas raised to 85° C. for 5.5 hours. The concentration of peptide in thesoluble fraction was 0.332 mg/mL.

EXAMPLE 5 Acid Hydrolysis of Bacteria After Removal of the Cell CultureMedium

E. coli E104 containing pING4703 was grown in a 14 L fermentor asdescribed in Example 2, and 10 mL of the fermentation culture wasadjusted to pH 2.2 with concentrated HCl. The sample was incubated at85° C., and samples were taken every few hours and analyzed for peptidein the supernatant by HPLC, using the same method as described inExample 3 for quantitation of peptide from the product encoded bypING4702. The results from this study are shown in the following Table8.

TABLE 8 Peptide Concentration Time at 85° C. mg/mL 4 0.018 6 0.011 70.004

These peptide titers were much lower than what was obtained withcultures of E. coli E104 (pING4702) as described in Examples 3 and 4,and lower than the titer obtained when E. coli E104 (pING4703) was lysedby sonication after incubation with lysozyme by the process described inExample 3. E. coli E104 (pING4703) lysed by sonication after lysozymetreatment had a titer of approximately 0.46 mg/mL in the solublefraction.

In additional studies, samples of the bacterial cell culture both beforeand after acid hydrolysis at 85° C. were analyzed by SDS-PAGE. Theresults demonstrated that the fusion protein of Bone D and peptide hadbeen hydrolyzed by acid. An experiment was executed to determine if thecell culture medium in the hydrolysis reaction had an impact on theability to recover recombinant peptide in the soluble fraction, since aprominent band at the position of Bone D was apparent in the hydrolyzedsample, while very little intact fusion protein was detected. Cell pastefrom the fermentation of E. coli E104 (pING4703) was prepared bycentrifugation, and 1 g of cell paste was suspended in: 7 mL H20; 7 mLof 5 mM EDTA; or 7 mL of cell-free fermentation broth from the samebacterial fermentor. Each sample was adjusted to pH 2.2 withconcentrated HCl, and incubated at 85° C. The amount of recombinantpeptide in the soluble fraction was measured over time by HPLC. Theresults are shown in the following Table 9.

TABLE 9 Medium Time (Hours) H₂O Sample 5 mM EDTA Sample Sample at 85° C.Peptide Concentration by HPLC (mg/mL) 1 0.052 0.043 0 2 0.184 0.1870.011 4 0.269 0.284 0.009 6 0.267 not determined 0.003 8 0.255 notdetermined not determined

These data demonstrated that the recombinant peptide was soluble whenthe cells were hydrolyzed in water or 5 mM EDTA, but did not becomesoluble in the fermentation medium after acid hydrolysis.

Further studies were performed to determine if recombinant peptide wasinsoluble in acid after hydrolysis from Bone D, and could be releasedfrom the insoluble material in detergents or chaotropic salts. Three 1gram samples of cell paste from E. coli E104 (pING4703) were suspendedin 7 ml of 100 mM Tris, 5 mM EDTA, pH 8.0, and one 1 gram sample of cellpaste was suspended in 7 mL of cell-free culture medium from the E. colifermentation. To one of the samples suspended in Tris buffer, 1 mL of 10mg/mL lysozyrne was added, the sample was incubated on ice and sonicatedas described in Example 3. The pH of all four samples was adjusted toapproximately pH 2.0 with concentrated HCl, and the samples wereincubated at 85° C. for 4 hours. By HPLC, the amount of peptide releasedinto the soluble fraction from the four samples was as follows in Table10.

TABLE 10 Peptide Concentration Sample Suspension buffer mg/mL 1 100 mMTris, 5 mM EDTA 0.594 2 100 mM Tris, 5 mM EDTA 0.618 3 Medium 0 4 100 mMTris, 5 mM EDTA + 0.519 Lysozyme and sonication

Thus, peptide did not appear in the soluble fraction in Sample 3 afteracid hydrolysis. To determine if peptide could be released from theinsoluble material, the pellet from Sample 3 was washed sequentiallywith 7 mL of buffer containing Triton X-100, urea, guanidinehydrochloride or SDS. The amount of peptide released from the pellet wasas follows in Table 11.

TABLE 11 Peptide concentration Total Peptide Released (mg/mL) in the mgpeptide/gram of Wash Buffer Wash Buffer cells 1% Triton X-100 in 10 mM0.01 0.08 sodium phosphate, pH 7.0 3% Triton S-100 in 10 mM 0 0 sodiumcitrate, pH 3.0 4M urea in 10 mM sodium 0.04 0.29 citrate, pH 3.0 8Murea in 10 mM sodium 0.08 0.55 citrate, pH 3.0 - first wash 8M urea in10 mM sodium 0.07 0.49 citrate, pH 3.0 - second wash 8M urea in 10 mMsodium 0.06 0.39 citrate, pH 3.0 third wash for 15 hours 6 M guanidine0.12 0.87 hydrochloride 4% SDS 0 0 Total in all washes: 2.67

These results demonstrated that the peptide could be recovered from theinsoluble material by washing in buffers containing urea or guanidinehydrochloride. The peptide was therefore not degraded by the hydrolysiscondition, but is rendered insoluble by media components. The totalamount of material recoverable in all washes was 2.67 mg per gram ofcells, compared to 4.16 mg/g and 3.63 mg/g recovered directly from thesoluble material in Samples 1 and 4 , respectively. Thus, for somebacterial cell cultures, the bacterial cells may be preferentiallyremoved from the media and the bacterial cells may be acidifiedaccording to Example 3. For other bacterial cell cultures, thefermentation broth (bacterial cells in cell culture/fermentation media)may be directly acidified according to Examples 3 and 4.

EXAMPLE 6 Recovery and Purification of Recombinant Peptide from AcidHydrolyzed Cells

1. Recovery

The invention provides methods for the recovery of peptides in thesoluble fraction after acid hydrolysis of cells while the large majorityof other bacterial proteins, the carrier protein, and other impuritiesremain in the insoluble fraction. The soluble and insoluble material canbe separated by centrifugation, filtration or any other suitableseparation method. Any variety of centrifuge can be used to separatethese materials and a variety of filtration devices, systems and methodscan also be used. A variety of such filtration devices, systems andmethods were used to separate soluble and insoluble materials includingdead end (depth) filtration and tangential flow filtration. A summary ofthe results of exemplary filtration studies to separate soluble andinsoluble material by filtration is presented in the following Table 12.

TABLE 12 Filtration Filter Through- Permeate Method Sample AnalyzedDescription put description Recovery Depth Previously frozen Seitz 9001.5 L Clear 72% cell paste SD/SDC, 1 suspended in ft², 8 μm water andacid nominal hydrolyzed retention Depth Previously frozen Seitz 5.5 LClear 94% preceded cell paste SD250, by 1 μm suspended in 4 1 ft², 4 μmbag filter volumes of water nominal and acid retention hydrolyzed DepthAcid hydrolysate Cuno Zeta 1.7 L Cloudy ND prepared directly Plus 01A, 1in a 35 L ft², 7 μm fermentor nominal retention Depth Acid hydrolysateCuno 30 SP, 1.2 L Cloudy ND preceded prepared directly 1 ft², 0.6 μm by1 μm in a 35 L nominal bag filter fermentor retention Depth Previouslyfrozen Cuno Zeta 27 mL Slightly ND with and cell paste Plus 01A, with nocloudy without suspended in 3.8 28 cm² Celite; Celite volumes ofwater >50 mL filter aid and acid with hydrolyzed Celite (HP³ 1000)Celite Acid hydrolysate Celite 2 L/min Clear 73% filter aid prepareddirectly (Hy-flo) in in a 35 L precoat, 600 horizontal fermentor cm²pressure leaf vessel Tangential Previously frozen Sartorius ND Cloudy NDFlow cell paste 0.2 cutoff suspended in 3.8 filter, 0.1 m² volumes ofwater and acid hydrolyzed Tangential Previously frozen 300 kDa 150 Clear81% Flow cell paste MWCO, mL/min suspended in 3.8 0.1 m² volumes ofwater and acid hydrolyzed ND-not determined Seitz filters are productsof SWK Filtration Incorporated, Petaluma, CA. Cuno filters are productsof Cuno Meriden, CT. Celite is a product of World Minerals, Lompoc, CA.Sartorius filters are products of Sartorius, Edgewood, NY.

These results demonstrate that a variety of filtration devices, systemsand methods can be successfully employed to separate the soluble andinsoluble material.

2. Purification

Following fermentation of E. coli E104 containing pING4702 as describedin Example 2, bacterial cells in the unprocessed fermentation broth werehydrolyzed in dilute HCl. Specifically, 40 mL of fermentation broth wasadjusted 15 to pH 2.15 with concentrated HCl. The sample was incubatedat 85° C. for 5.5 hours. The hydrolyzed cells were then centrifuged toremove insoluble material, and the supernatant was adjusted to pH 3.0 byadding 500 mM sodium citrate dropwise.

An SP Sepharose (Amersham-Pharmacia, Piscataway, N.J.) column, 2.5×4.4cm containing 21.6 mL, was equilibrated in 10 mM sodium citrate, pH 3.0and the sample was loaded. The column was washed with 10 mM sodiumcitrate, pH 3.0 buffer and then 10 mM sodium phosphate, pH 7.0 until thepH of the column effluent reached 7. The column was then washed in 10 mMsodium phosphate, 150 mM NaCl pH 7.0. The column was eluted in 10 mMsodium phosphate, 800 mM NaCl pH 7.0 and then the column was strippedwith 10 mM sodium phosphate, 2 M NaCl. The SP Sepharose eluate wasdiluted with one volume of 10 mM sodium phosphate, 3 M ammonium sulfate,pH 7.0.

A Butyl Sepharose (Amersham-Pharmacia) column, 1×4 cm containing 3.1 mL,was equilibrated with 10 mM sodium phosphate, 1.5 M ammonium sulfate, pH7.0, and the sample was loaded. The Butyl Sepharose column was washedwith 10 mM sodium phosphate, 1.1 M ammonium sulfate, pH 7.0, and theneluted with 10 mM sodium phosphate, 0.4 M ammonium sulfate, pH 7.0. Thecolumn was striped with 10 mM sodium phosphate, pH 7.0

The peptide concentration in each of the fractions from the SP Sepharoseand Butyl Sepharose columns was followed by HPLC analysis. The samplevolumes, peptide concentrations and percent recovery was as follows inTable 13.

TABLE 13 Volume Concentration Total Sample (mL) (mg/mL) mg % Yield SPSepharose load 24 0.399 9.58 100 First SP Sepharose wash 75 0 0 0 SecondSP Sepharose wash 33 0 0 0 SP Sepharose eluate 50 0.165 8.25 86.1 SPSepharose strip 13 0.036 0.47 4.9 Butyl Sepharose load 94 ND ND ND ButylSepharose flow 95 0 0 0 through Butyl Sepharose wash 13 0.008 0.1 1Butyl Sepharose eluate 28 0.262 7.34 76.6 Butyl Sepharose strip 4 0.0140.06 0.6 ND-Not Determined

A Superdex 30 (Amersham-Pharmacia) column, 1.6×53 cm containing 107 mL,was equilibrated in 5 mM sodium acetate, 150 mM NaCl, pH 5.0. Eight mLof Butyl Sepharose eluate was loaded onto the Superdex 30 gel filtrationcolumn, and the column was run with 5 mM sodium acetate, 150 mM NaCl, pH5.0. After 32 mL had flowed through the column, 3 mL fractions werecollected. Fractions 12-19 were pooled and had a volume of approximately20 mL. The concentration of recombinant peptide in the Superdex 30 poolwas 0.107 mg/mL for a recovery of 102% from the previous step, and theoverall recovery from the acid hydrolysate of cells was 76.6%. The finalpeptide purity was 97.4%.

EXAMPLE 7 Biological Activity Assays of Recombinant Peptides

A variety of recombinant peptides, including those BPI-derived peptidescomprising the sequences listed in U.S. Pat. No. 5,851,802 incorporatedby reference, may be produced by recombinant methods of the inventionand tested for biological activity by known activity assays. Assays forantimicrobial activity (both anti-fungal and anti-bacterial activity)may be performed, including radial diffusion assays. Assays, with avariety of fungal and bacterial cells, including those described in U.S.Pat. No. 5,851,802 (see Example 6), may be conducted using recombinantpeptides produced according to the invention.

For example, studies were performed to evaluate the antifungal activityof the recombinant peptide from pING4702 purified according to Example 6in a broth microdilution assay using four strains of C. albicans, C.glabrata and S. cerevisiae. A similar peptide, XMP.391, that waschemically synthesized, was included in the assay as a positive control.To perform the broth microdilution assay, the fungal cultures were grownovernight at 30° C. in YPD medium (1% yeast extract, 2% peptone, and 2%dextrose). A 400 fold dilution of each culture in YPD was then made, andgrown at 30° C. for 8 hours. Three mL of each culture were collected bycentrifugation and suspended in 0.9% NaCl to an A600 of about 0.3. Thesecultures were further diluted to 1×10⁴ CFU/mL in Sabouraud dextrosebroth (6 mL). Recombinant peptide was in 5 mM acetate, 150 mM NaCl, pH5.0 at a concentration of about 2 mg/mL. Synthetic peptide was at about1 mg/mL. Samples were serially diluted and added to microtiter platescontaining the cultures. Plates were incubated at 30° C. for 48 hoursbefore growth inhibition was measured. Results from this assay were asfollows in Table 14.

TABLE 14 Synthetic Peptide XMP.391 Recombinant Peptide (Concentrationthat gives (Concentration that gives Strain 95% inhibition (μM)) 95%inhibition (μM)) C. albicans SLU1 13.5 16 C. albicans 10231 16 30 C.albicans 14053 16 16 C. albicans 26555 16 30 C. glabrata 2001 30 30 S.cerevisiae 9763 2.0 7.5

Additionally or alternatively, assays may be performed to assess theendotoxin binding and neutralizing activity of the recombinantlyproduced peptides, by a variety of known assays, including thosedescribed in co-owned U.S. Pat. Nos. 5,733,872 and 5,763,567 [WO94/20532 (PCT/US94/02465)]; U.S. Pat. Nos. 5,652,332 and 5,856,438 [WO95/19372 (PCT/US94/10427)]; U.S. Pat. No. 5,858,974 [WO 96/08509(PCT/US95/09262) and WO 97/04008 PCT/US96/03845)]; incorporated byreference in their entirety.

Additionally or alternatively, assays may be performed to assess theheparin binding and neutralizing activity of the recombinantly producedpeptides by a variety of known assays, including assays as described inU.S. Pat. Nos. 5,348,942; 5,639,727; 5,807,818; 5,837,678; and 5,854,214[WO 94/20128 (PCT/US94/02401)]; U.S. Pat. Nos. 5,733,872 and 5,763,567[WO 94/20532 (PCT/US94/02465)]; U.S. Pat. Nos. 5,652,332 and 5,856,438[WO 95/19372 PCT/US94/10427)]; incorporated by reference in theirentirety.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims. In particular, numerousmodifications and variations in the practice of the invention areexpected to occur to those skilled in the art upon consideration of theforegoing description on the presently preferred embodiments thereof.Consequently, the only limitations which should be placed upon the scopeof the present invention are those that appear in the appended claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1 <211>LENGTH: 557 <212> TYPE: DNA <213> ORGANISM: Human <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (66)..(548) <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(65) <223> OTHER INFORMATION: /label= EcoRI/note=“residues 1-65 comprise EcoRI site to beginning of pel B.” <221>NAME/KEY: misc_feature <222> LOCATION: (1)..(22) <223> OTHERINFORMATION: /label=pel B /note=“pel B is the leader sequence from thepectate lyase gene of Erwinia carotovora.” <221> NAME/KEY: misc_feature<222> LOCATION: (23)..(161) <223> OTHER INFORMATION: /label=“Bone D”/note=“Bone D is the subunit of human osteogenic protein (see, U.S.Patent No. 5,284,756 e.g., Fig. 6, Example 9, Seq ID Nos: 1 and 2.”<221> NAME/KEY: misc_feature <222> LOCATION: (549)..(557) <223> OTHERINFORMATION: /label=XHoI /note=“residues 549-557 comprise stop codon andXhoI site.” <400> SEQUENCE: 1 gaattcctgc aggtctatgg aacgataaatgcccatgaaa attctatttc aaggagacag 60 tcata atg aaa tac cta ttg cct acggca gcc gct gga ttg tta tta ctc 110 Met Lys Tyr Leu Leu Pro Thr Ala AlaAla Gly Leu Leu Leu Leu 1 5 10 15 gct gcc caa cca gcg atg gcg tcc acgggg agc aaa cag cgc agc cag 158 Ala Ala Gln Pro Ala Met Ala Ser Thr GlySer Lys Gln Arg Ser Gln 20 25 30 aac cgc tcc aag acg ccc aag aac cag gaagcc ctg cgg atg gcc aac 206 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu AlaLeu Arg Met Ala Asn 35 40 45 gtg gca gag aac agc agc agc gac cag agg caggcc tgt aag aag cac 254 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln AlaCys Lys Lys His 50 55 60 gag ctg tat gtc agc ttc cga gac ctg ggc tgg caggac tgg atc atc 302 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln AspTrp Ile Ile 65 70 75 gcg cct gaa ggc tac gcc gcc tac tac tgt gag ggg gagtgt gcc ttc 350 Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu CysAla Phe 80 85 90 95 cct ctg aac tcc tac atg aac gcc acc aac cac gcc atcgtg cag acg 398 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile ValGln Thr 100 105 110 ctg gtc cac ttc atc aac ccg gaa acg gtg ccc aag ccctgc tgt gcg 446 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro CysCys Ala 115 120 125 ccc acg cag ctc aat gcc atc tcc gtc ctc tac ttc gatgac agc tcc 494 Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp AspSer Ser 130 135 140 aac gtc atc ctg aag aaa tac aga aac atg gtg gtc cgggcc tgt ggc 542 Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg AlaCys Gly 145 150 155 tgc cac tagctcgag 557 Cys His 160 <210> SEQ ID NO 2<211> LENGTH: 161 <212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE:2 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 1015 Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 20 2530 Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 35 4045 Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 50 5560 Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 65 7075 80 Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 8590 95 Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu100 105 110 Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys AlaPro 115 120 125 Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp SerSer Asn 130 135 140 Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg AlaCys Gly Cys 145 150 155 160 His <210> SEQ ID NO 3 <211> LENGTH: 12 <212>TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 3 Lys Val Gly Trp LeuIle Gln Leu Phe His Lys Lys 1 5 10 <210> SEQ ID NO 4 <211> LENGTH: 51<212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 4 gatccaccgaaagtgggttg gctgatccag ctgttccaca aaaagtaaag c 51 <210> SEQ ID NO 5 <211>LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 5tcgagcttta ctttttgtgg aacagctgga tcagccaacc cactttcggt g 51 <210> SEQ IDNO 6 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Human <400>SEQUENCE: 6 Asp Pro Pro Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys1 5 10 15 <210> SEQ ID NO 7 <211> LENGTH: 610 <212> TYPE: DNA <213>ORGANISM: Human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(66)..(599) <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(65) <223>OTHER INFORMATION: /label=EcoRI /note=“residues 1-65 comprise EcoRI siteto beginning of pel B.” <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(22) <223> OTHER INFORMATION: /label=pel B /note=“pel B is theleader sequence from the pectate lyase gene of Erwinia caratovora.”<221> NAME/KEY: misc_feature <222> LOCATION: (23)..(161) <223> OTHERINFORMATION: /label=“Bone D” /note=“Bone D is the subunit of humanosteogenic protein (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example9, Seq ID Nos: 1 and 2.” <221> NAME/KEY: misc_feature <222> LOCATION:(162)..(166) <223> OTHER INFORMATION: /label=cleavage linker/note=“Ala-Leu-Asp-Pro- Pro linking sequence with Asp-Pro cleavagesite.” <221> NAME/KEY: misc_feature <222> LOCATION: (167)..(178) <223>OTHER INFORMATION: /label=peptide sequence /note=“BPI-derived peptide.”<221> NAME/KEY: misc_feature <222> LOCATION: (600)..(610) <223> OTHERINFORMATION: /label=XhoI /note=“residues 600-610 comprise stop codon andXhoI site.” <400> SEQUENCE: 7 gaattcctgc aggtctatgg aacgataaatgcccatgaaa attctatttc aaggagacag 60 tcata atg aaa tac cta ttg cct acggca gcc gct gga ttg tta tta ctc 110 Met Lys Tyr Leu Leu Pro Thr Ala AlaAla Gly Leu Leu Leu Leu 1 5 10 15 gct gcc caa cca gcg atg gcg tcc acgggg agc aaa cag cgc agc cag 158 Ala Ala Gln Pro Ala Met Ala Ser Thr GlySer Lys Gln Arg Ser Gln 20 25 30 aac cgc tcc aag acg ccc aag aac cag gaagcc ctg cgg atg gcc aac 206 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu AlaLeu Arg Met Ala Asn 35 40 45 gtg gca gag aac agc agc agc gac cag agg caggcc tgt aag aag cac 254 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln AlaCys Lys Lys His 50 55 60 gag ctg tat gtc agc ttc cga gac ctg ggc tgg caggac tgg atc atc 302 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln AspTrp Ile Ile 65 70 75 gcg cct gaa ggc tac gcc gcc tac tac tgt gag ggg gagtgt gcc ttc 350 Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu CysAla Phe 80 85 90 95 cct ctg aac tcc tac atg aac gcc acc aac cac gcc atcgtg cag acg 398 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile ValGln Thr 100 105 110 ctg gtc cac ttc atc aac ccg gaa acg gtg ccc aag ccctgc tgt gcg 446 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro CysCys Ala 115 120 125 ccc acg cag ctc aat gcc atc tcc gtc ctc tac ttc gatgac agc tcc 494 Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp AspSer Ser 130 135 140 aac gtc atc ctg aag aaa tac aga aac atg gtg gtc cgggcc tgt ggc 542 Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg AlaCys Gly 145 150 155 tgc cac gca ttg gat cca ccg aaa gtg ggt tgg ctg atccag ctg ttt 590 Cys His Ala Leu Asp Pro Pro Lys Val Gly Trp Leu Ile GlnLeu Phe 160 165 170 175 cat aaa aag taaagctcga g 610 His Lys Lys <210>SEQ ID NO 8 <211> LENGTH: 178 <212> TYPE: PRT <213> ORGANISM: Human<400> SEQUENCE: 8 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu LeuLeu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys GlnArg Ser Gln Asn 20 25 30 Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu ArgMet Ala Asn Val 35 40 45 Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala CysLys Lys His Glu 50 55 60 Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln AspTrp Ile Ile Ala 65 70 75 80 Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu GlyGlu Cys Ala Phe Pro 85 90 95 Leu Asn Ser Tyr Met Asn Ala Thr Asn His AlaIle Val Gln Thr Leu 100 105 110 Val His Phe Ile Asn Pro Glu Thr Val ProLys Pro Cys Cys Ala Pro 115 120 125 Thr Gln Leu Asn Ala Ile Ser Val LeuTyr Phe Asp Asp Ser Ser Asn 130 135 140 Val Ile Leu Lys Lys Tyr Arg AsnMet Val Val Arg Ala Cys Gly Cys 145 150 155 160 His Ala Leu Asp Pro ProLys Val Gly Trp Leu Ile Gln Leu Phe His 165 170 175 Lys Lys <210> SEQ IDNO 9 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Human <400>SEQUENCE: 9 Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys Val GlyTrp 1 5 10 15 Leu Ile Leu Leu Phe His Lys Lys 20 <210> SEQ ID NO 10<211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE:10 cattggatcc accgaaatgg aaggcccagt ttcgctttct taagaaatcg aaagtgggtt 60g 61 <210> SEQ ID NO 11 <211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM:Human <400> SEQUENCE: 11 ggctctcgag ctctactttt tatgaaacag caggatcagccaacccactt tcgatttctt 60 a 61 <210> SEQ ID NO 12 <211> LENGTH: 27 <212>TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 12 Asp Pro Pro Lys TrpLys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys 1 5 10 15 Val Gly Trp LeuIle Leu Leu Phe His Lys Lys 20 25 <210> SEQ ID NO 13 <211> LENGTH: 646<212> TYPE: DNA <213> ORGANISM: Human <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (66)..(635) <221> NAME/KEY: misc_feature <222> LOCATION:(1)..(65) <223> OTHER INFORMATION: /label= EcoRI /note=“residues 1-65comprise EcoRI site to beginning of pel B.” <221> NAME/KEY: misc_feature<222> LOCATION: (1)..(22) <223> OTHER INFORMATION: /label=pel B/note=“pel B is the leader sequence from the pectate lyase gene ofErwinia caratovora.” <221> NAME/KEY: misc_feature <222> LOCATION:(23)..(161) <223> OTHER INFORMATION: /label= “Bone D” /note=“Bone D isthe subunit of human osteogenic protein (see, U.S. Patent No. 5,284,756e.g., Fig. 6, Example 9, Seq ID NOs: 1 and 2.′ <221> NAME/KEY:misc_feature <222> LOCATION: (162)..(166) <223> OTHER INFORMATION:/label=cleavage linker /note=”Ala-Leu-Asp-Pro- Pro linking sequence withAsp-Pro cleavage site.“ <221> NAME/KEY: misc_feature <222> LOCATION:(167)..(190) <223> OTHER INFORMATION: /label=peptide sequence/note=”BPI-derived peptide.“ <221> NAME/KEY: misc_feature <222>LOCATION: (636)..(646) <223> OTHER INFORMATION: /label=XhoI/note=”residues 636-646 comprise stop codon and XhoI site.“ <400>SEQUENCE: 13 gaattcctgc aggtctatgg aacgataaat gcccatgaaa attctatttcaaggagacag 60 tcata atg aaa tac cta ttg cct acg gca gcc gct gga ttg ttatta ctc 110 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu1 5 10 15 gct gcc caa cca gcg atg gcg tcc acg ggg agc aaa cag cgc agccag 158 Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln20 25 30 aac cgc tcc aag acg ccc aag aac cag gaa gcc ctg cgg atg gcc aac206 Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn 3540 45 gtg gca gag aac agc agc agc gac cag agg cag gcc tgt aag aag cac254 Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His 5055 60 gag ctg tat gtc agc ttc cga gac ctg ggc tgg cag gac tgg atc atc302 Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile 6570 75 gcg cct gaa ggc tac gcc gcc tac tac tgt gag ggg gag tgt gcc ttc350 Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe 8085 90 95 cct ctg aac tcc tac atg aac gcc acc aac cac gcc atc gtg cag acg398 Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr 100105 110 ctg gtc cac ttc atc aac ccg gaa acg gtg ccc aag ccc tgc tgt gcg446 Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala 115120 125 ccc acg cag ctc aat gcc atc tcc gtc ctc tac ttc gat gac agc tcc494 Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser 130135 140 aac gtc atc ctg aag aaa tac aga aac atg gtg gtc cgg gcc tgt ggc542 Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly 145150 155 tgc cac gca ttg gat cca ccg aaa tgg aag gcc cag ttt cgc ttt ctt590 Cys His Ala Leu Asp Pro Pro Lys Trp Lys Ala Gln Phe Arg Phe Leu 160165 170 175 aag aaa tcg aaa gtg ggt tgg ctg atc ctg ctg ttt cat aaa aag635 Lys Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 180 185190 tagagctcga g 646 <210> SEQ ID NO 14 <211> LENGTH: 190 <212> TYPE:PRT <213> ORGANISM: Human <400> SEQUENCE: 14 Met Lys Tyr Leu Leu Pro ThrAla Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met AlaSer Thr Gly Ser Lys Gln Arg Ser Gln Asn 20 25 30 Arg Ser Lys Thr Pro LysAsn Gln Glu Ala Leu Arg Met Ala Asn Val 35 40 45 Ala Glu Asn Ser Ser SerAsp Gln Arg Gln Ala Cys Lys Lys His Glu 50 55 60 Leu Tyr Val Ser Phe ArgAsp Leu Gly Trp Gln Asp Trp Ile Ile Ala 65 70 75 80 Pro Glu Gly Tyr AlaAla Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 85 90 95 Leu Asn Ser Tyr MetAsn Ala Thr Asn His Ala Ile Val Gln Thr Leu 100 105 110 Val His Phe IleAsn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 115 120 125 Thr Gln LeuAsn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 130 135 140 Val IleLeu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 145 150 155 160His Ala Leu Asp Pro Pro Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys 165 170175 Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 180 185 190<210> SEQ ID NO 15 <211> LENGTH: 1813 <212> TYPE: DNA <213> ORGANISM:Human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (31)..(1491)<221> NAME/KEY: mat_peptide <222> LOCATION: (124)..(1491) <223> OTHERINFORMATION: ”rBPI“ <400> SEQUENCE: 15 caggccttga ggttttggca gctctggaggatg aga gag aac atg gcc agg ggc 54 Met Arg Glu Asn Met Ala Arg Gly -30-25 cct tgc aac gcg ccg aga tgg gtg tcc ctg atg gtg ctc gtc gcc ata 102Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile -20 -15-10 ggc acc gcc gtg aca gcg gcc gtc aac cct ggc gtc gtg gtc agg atc 150Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile -5 -1 15 tcc cag aag ggc ctg gac tac gcc agc cag cag ggg acg gcc gct ctg 198Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu 10 15 2025 cag aag gag ctg aag agg atc aag att cct gac tac tca gac agc ttt 246Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser Phe 30 35 40aag atc aag cat ctt ggg aag ggg cat tat agc ttc tac agc atg gac 294 LysIle Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp 45 50 55 atccgt gaa ttc cag ctt ccc agt tcc cag ata agc atg gtg ccc aat 342 Ile ArgGlu Phe Gln Leu Pro Ser Ser Gln Ile Ser Met Val Pro Asn 60 65 70 gtg ggcctt aag ttc tcc atc agc aac gcc aat atc aag atc agc ggg 390 Val Gly LeuLys Phe Ser Ile Ser Asn Ala Asn Ile Lys Ile Ser Gly 75 80 85 aaa tgg aaggca caa aag aga ttc tta aaa atg agc ggc aat ttt gac 438 Lys Trp Lys AlaGln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 90 95 100 105 ctg agcata gaa ggc atg tcc att tcg gct gat ctg aag ctg ggc agt 486 Leu Ser IleGlu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser 110 115 120 aac cccacg tca ggc aag ccc acc atc acc tgc tcc agc tgc agc agc 534 Asn Pro ThrSer Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser 125 130 135 cac atcaac agt gtc cac gtg cac atc tca aag agc aaa gtc ggg tgg 582 His Ile AsnSer Val His Val His Ile Ser Lys Ser Lys Val Gly Trp 140 145 150 ctg atccaa ctc ttc cac aaa aaa att gag tct gcg ctt cga aac aag 630 Leu Ile GlnLeu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys 155 160 165 atg aacagc cag gtc tgc gag aaa gtg acc aat tct gta tcc tcc aag 678 Met Asn SerGln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys 170 175 180 185 ctgcaa cct tat ttc cag act ctg cca gta atg acc aaa ata gat tct 726 Leu GlnPro Tyr Phe Gln Thr Leu Pro Val Met Thr Lys Ile Asp Ser 190 195 200 gtggct gga atc aac tat ggt ctg gtg gca cct cca gca acc acg gct 774 Val AlaGly Ile Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala 205 210 215 gagacc ctg gat gta cag atg aag ggg gag ttt tac agt gag aac cac 822 Glu ThrLeu Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 cacaat cca cct ccc ttt gct cca cca gtg atg gag ttt ccc gct gcc 870 His AsnPro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala 235 240 245 catgac cgc atg gta tac ctg ggc ctc tca gac tac ttc ttc aac aca 918 His AspArg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 250 255 260 265gcc ggg ctt gta tac caa gag gct ggg gtc ttg aag atg acc ctt aga 966 AlaGly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met Thr Leu Arg 270 275 280gat gac atg att cca aag gag tcc aaa ttt cga ctg aca acc aag ttc 1014 AspAsp Met Ile Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe 285 290 295ttt gga acc ttc cta cct gag gtg gcc aag aag ttt ccc aac atg aag 1062 PheGly Thr Phe Leu Pro Glu Val Ala Lys Lys Phe Pro Asn Met Lys 300 305 310ata cag atc cat gtc tca gcc tcc acc ccg cca cac ctg tct gtg cag 1110 IleGln Ile His Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln 315 320 325ccc acc ggc ctt acc ttc tac cct gcc gtg gat gtc cag gcc ttt gcc 1158 ProThr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala 330 335 340345 gtc ctc ccc aac tcc tcc ctg gct tcc ctc ttc ctg att ggc atg cac 1206Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly Met His 350 355360 aca act ggt tcc atg gag gtc agc gcc gag tcc aac agg ctt gtt gga 1254Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly 365 370375 gag ctc aag ctg gat agg ctg ctc ctg gaa ctg aag cac tca aat att 1302Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys His Ser Asn Ile 380 385390 ggc ccc ttc ccg gtt gaa ttg ctg cag gat atc atg aac tac att gta 1350Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Ile Met Asn Tyr Ile Val 395 400405 ccc att ctt gtg ctg ccc agg gtt aac gag aaa cta cag aaa ggc ttc 1398Pro Ile Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 410 415420 425 cct ctc ccg acg ccg gcc aga gtc cag ctc tac aac gta gtg ctt cag1446 Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln 430435 440 cct cac cag aac ttc ctg ctg ttc ggt gca gac gtt gtc tat aaa 1491Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys 445 450 455tgaaggcacc aggggtgccg ggggctgtca gccgcacctg ttcctgatgg gctgtggggc 1551accggctgcc tttccccagg gaatcctctc cagatcttaa ccaagagccc cttgcaaact 1611tcttcgactc agattcagaa atgatctaaa cacgaggaaa cattattcat tggaaaagtg 1671catggtgtgt attttaggga ttatgagctt ctttcaaggg ctaaggctgc agagatattt 1731cctccaggaa tcgtgtttca attgtaacca agaaatttcc atttgtgctt catgaaaaaa 1791aacttctggt ttttttcatg tg 1813 <210> SEQ ID NO 16 <211> LENGTH: 487 <212>TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 16 Met Arg Glu Asn MetAla Arg Gly Pro Cys Asn Ala Pro Arg Trp Val -30 -25 -20 Ser Leu Met ValLeu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val -15 -10 -5 -1 1 Asn ProGly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala 5 10 15 Ser GlnGln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys 20 25 30 Ile ProAsp Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly 35 40 45 His TyrSer Phe Tyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser 50 55 60 65 SerGln Ile Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser 70 75 80 AsnAla Asn Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe 85 90 95 LeuLys Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile 100 105 110Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120125 Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His 130135 140 145 Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His LysLys 150 155 160 Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Ser Gln Val CysGlu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr PheGln Thr Leu 180 185 190 Pro Val Met Thr Lys Ile Asp Ser Val Ala Gly IleAsn Tyr Gly Leu 195 200 205 Val Ala Pro Pro Ala Thr Thr Ala Glu Thr LeuAsp Val Gln Met Lys 210 215 220 225 Gly Glu Phe Tyr Ser Glu Asn His HisAsn Pro Pro Pro Phe Ala Pro 230 235 240 Pro Val Met Glu Phe Pro Ala AlaHis Asp Arg Met Val Tyr Leu Gly 245 250 255 Leu Ser Asp Tyr Phe Phe AsnThr Ala Gly Leu Val Tyr Gln Glu Ala 260 265 270 Gly Val Leu Lys Met ThrLeu Arg Asp Asp Met Ile Pro Lys Glu Ser 275 280 285 Lys Phe Arg Leu ThrThr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val 290 295 300 305 Ala Lys LysPhe Pro Asn Met Lys Ile Gln Ile His Val Ser Ala Ser 310 315 320 Thr ProPro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 AlaVal Asp Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 340 345 350Ser Leu Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360365 Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370375 380 385 Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu LeuLeu 390 395 400 Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val Leu ProArg Val 405 410 415 Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr ProAla Arg Val 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln AsnPhe Leu Leu Phe 435 440 445 Gly Ala Asp Val Val Tyr Lys 450 455

What is claimed is:
 1. In a method for obtaining a peptide from bacterial cells after expression inside the cells of a fusion protein, wherein the fusion protein comprises the peptide, a carrier protein and an acid-cleavable site between the peptide and the carrier protein, the improvement comprising: treating the bacterial cells with acid under conditions sufficient in a single step to disrupt or lyse the cells and release the peptide from the fusion protein, wherein the bacterial cells are in cell culture media for the acid treatment.
 2. The method of claim 1 with the additional step of obtaining the released peptide separated from the disrupted or lysed cells.
 3. The method of claim 2 wherein the released peptide is separated from the disrupted or lysed cells by a separation device.
 4. The method of claim 3 wherein the separation device is a centrifugation device.
 5. The method of claim 3 wherein the separation device is a filtration device.
 6. The method of claim 1 wherein the acid-cleavable site in the fusion protein is Asp-Pro.
 7. The method of claim 1 wherein the carrier protein is expressed as an insoluble protein inside the bacterial cells.
 8. The method of claim 7 wherein the carrier protein is the D subunit of human osteogenic protein.
 9. The method of claim 1 wherein the bacterial cells have been separated from cell culture media for the acid treatment.
 10. The method of claim 1 wherein the bacterial cells are in cell culture media in a fermentation vessel for the acid treatment.
 11. In a method for obtaining a peptide from bacterial cells after expression inside the cells of a fusion protein, wherein the fusion protein comprises the peptide, a carrier protein and an acid-cleavable site between the peptide and the carrier protein, the improvement comprising: (a) treating the bacterial cells with acid under conditions sufficient to disrupt or lyse the cells and release the peptide from the fusion protein, wherein the bacterial cells are in cell culture media for the acid treatment, (b) separating soluble material from insoluble material after step (a), and (c) recovering the released peptide in the soluble material after step (b).
 12. The method of claim 11 wherein the soluble material is separated from the insoluble material by a separation device.
 13. The method of claim 12 wherein the separation device is a centrifugation device.
 14. The method of claim 12 wherein the separation device is a filtration device.
 15. The method of claim 11 wherein the acid-cleavable site in the fusion protein is Asp-Pro.
 16. The method of claim 11 wherein the carrier protein is expressed as an insoluble protein inside the bacterial cells.
 17. The method of claim 16 wherein the carrier protein is the D subunit of human osteogenic protein.
 18. The method of claim 11 wherein the bacterial cells have been separated from cell culture media for the acid treatment.
 19. The method of claim 11 wherein the bacterial cells are in cell culture media in a fermentation vessel for the acid treatment. 